9
1 online | memorias.ioc.fiocruz.br Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(1): 1-8, February 2011 Culex Linnaeus is the largest genus of the Culicine with 768 species subdivided into 26 subgenera (Harbach 2011). The Culex genus includes species suspected of be- ing potential vectors for filarial worms and several arbovi- ruses, including the Venezuelan equine encephalitis virus (Weaver et al. 2004) and the West Nile virus (Turell et al. 2001). In the Americas, members of this extensive genus are subdivided into 14 subgenera. The subgenera Melano- conion Theobald and Culex Linnaeus are those of greatest epidemiological importance in the region and include most of the neotropical species. In Brazil, the Saint Louis en- cephalitis virus has been isolated from Culex ( Culex ) coro- nator Dyar and Knab and Culex ( Cux.) declarator Dyar and Knab (Vasconcelos et al. 1991). Culex ( Cux. ) quinque- fasciatus Say is the main vector for Wuchereria bancrofti, with active transmission in areas in the Northeast of Brazil (Fontes et al. 1998, 2005). Despite the medical importance of some species of the genus Culex and the great discom- fort they cause, little progress has been made in the tax- onomy and systematics of the genus. Most Culex mosquito research focuses on the epidemiology of arboviruses and potential vectors (Turell et al. 2001, Weaver et al. 2004) and on the control of species that transmit pathogens to humans and other vertebrates (Regis et al. 1995, Melo et al. 2009). The taxonomic history of Culex demonstrates the complexity of the group and the difficulty in differenti- ating its species using traditional morphological criteria alone. The morphological identification of these mos- quitoes is difficult because many species of Culex are morphologically similar (Bram 1967, Williams & Sav- age 2009). Furthermore, some Culex species belong to species complexes (Smith & Fonseca 2004), there are hybrid forms (Humeres et al. 1998, Smith & Fonseca 2004) and intraspecific variations in established diag- nostic characteristics are frequently reported (Rey et al. 2006). Lastly, field-collected adult specimens often lack ornamentation characteristics, making their identifica- tion difficult based on morphology alone (Knight & Na- yar 2004). There are few taxonomic studies for groups of Culex. In Bram’s (1967) revision of neotropical species of Culex and the subgenus Culex, a description of a new species, a re-description of several species and identifi- cation keys for the male genitalia and fourth instar larval characteristics are included. In another study, Tanaka (2003) elevated the subgenus Lutzia Theobald to the sta- tus of genus of the Culicine tribe. Accurate species identification is fundamental for ecological, biological and epidemiological studies. Di- agnosis based on traditional methods, including the external structures of the male genitalia, allows for initial identification that can be further tested using molecular techniques. Thus, many species previously considered unique and homogeneous turn out to consist of other isomorphic forms (Forattini 2002). Ribosomal DNA (rDNA) sequences have been used widely in mos- quito taxonomy and phylogenetic analyses (Walton et al. 1999, Sallum et al. 2002, Marrelli et al. 2005). Due Financial support: FAPESP (05/50225-2 for MTM and 05/53973-0), CNPq (BPP 300351/2008-9 to MAMS) FTV was a Master’s student supported by CAPES. + Corresponding author: [email protected] Received 23 April 2010 Accepted 4 November 2010 Intragenomic variation in the second internal transcribed spacer of the ribosomal DNA of species of the genera Culex and Lutzia (Diptera: Culicidae) Fabiana Tavares Vesgueiro 1 , Bruna Demari-Silva 1 , Rosely dos Santos Malafronte 2 , Maria Anice Mureb Sallum 1 , Mauro Toledo Marrelli 1 / + 1 Departamento de Epidemiologia, Faculdade de Saúde Pública 2 Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, São Paulo, SP, Brasil Culex is the largest genus of Culicini and includes vectors of several arboviruses and filarial worms. Many species of Culex are morphologically similar, which makes their identification difficult, particularly when using female speci- mens. To aid evolutionary studies and species distinction, molecular techniques are often used. Sequences of the sec- ond internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) from 16 species of the genus Culex and one of Lutzia were used to assess their genomic variability and to verify their applicability in the phylogenetic analysis of the group. The distance matrix (uncorrected p-distance) that was obtained revealed intragenomic and intraspecific variation. Because of the intragenomic variability, we selected ITS2 copies for use in distance analyses based on their second- ary structures. Neighbour-joining topology was obtained with an uncorrected p-distance. Despite the heterogeneity observed, individuals of the same species were grouped together and correlated with the current, morphology-based classification, thereby showing that ITS2 is an appropriate marker to be used in the taxonomy of Culex. Key words: Diptera - Culex - ITS2 - rDNA - phylogeny - concerted evolution

Intragenomic variation in the second internal transcribed ......Aedes (Aedimorphus) vexans Meigen (EF539857) and Aedes (Stegomyia) aegypti Linnaeus (AY512670). Be-cause of the large

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    online | memorias.ioc.fiocruz.br

    Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(1): 1-8, February 2011

    Culex Linnaeus is the largest genus of the Culicine with 768 species subdivided into 26 subgenera (Harbach 2011). The Culex genus includes species suspected of be-ing potential vectors for filarial worms and several arbovi-ruses, including the Venezuelan equine encephalitis virus (Weaver et al. 2004) and the West Nile virus (Turell et al. 2001). In the Americas, members of this extensive genus are subdivided into 14 subgenera. The subgenera Melano-conion Theobald and Culex Linnaeus are those of greatest epidemiological importance in the region and include most of the neotropical species. In Brazil, the Saint Louis en-cephalitis virus has been isolated from Culex (Culex) coro-nator Dyar and Knab and Culex (Cux.) declarator Dyar and Knab (Vasconcelos et al. 1991). Culex (Cux.) quinque-fasciatus Say is the main vector for Wuchereria bancrofti, with active transmission in areas in the Northeast of Brazil (Fontes et al. 1998, 2005). Despite the medical importance of some species of the genus Culex and the great discom-fort they cause, little progress has been made in the tax-onomy and systematics of the genus. Most Culex mosquito research focuses on the epidemiology of arboviruses and potential vectors (Turell et al. 2001, Weaver et al. 2004) and on the control of species that transmit pathogens to humans

    and other vertebrates (Regis et al. 1995, Melo et al. 2009). The taxonomic history of Culex demonstrates the

    complexity of the group and the difficulty in differenti-ating its species using traditional morphological criteria alone. The morphological identification of these mos-quitoes is difficult because many species of Culex are morphologically similar (Bram 1967, Williams & Sav-age 2009). Furthermore, some Culex species belong to species complexes (Smith & Fonseca 2004), there are hybrid forms (Humeres et al. 1998, Smith & Fonseca 2004) and intraspecific variations in established diag-nostic characteristics are frequently reported (Rey et al. 2006). Lastly, field-collected adult specimens often lack ornamentation characteristics, making their identifica-tion difficult based on morphology alone (Knight & Na-yar 2004). There are few taxonomic studies for groups of Culex. In Bram’s (1967) revision of neotropical species of Culex and the subgenus Culex, a description of a new species, a re-description of several species and identifi-cation keys for the male genitalia and fourth instar larval characteristics are included. In another study, Tanaka (2003) elevated the subgenus Lutzia Theobald to the sta-tus of genus of the Culicine tribe.

    Accurate species identification is fundamental for ecological, biological and epidemiological studies. Di-agnosis based on traditional methods, including the external structures of the male genitalia, allows for initial identification that can be further tested using molecular techniques. Thus, many species previously considered unique and homogeneous turn out to consist of other isomorphic forms (Forattini 2002). Ribosomal DNA (rDNA) sequences have been used widely in mos-quito taxonomy and phylogenetic analyses (Walton et al. 1999, Sallum et al. 2002, Marrelli et al. 2005). Due

    Financial support: FAPESP (05/50225-2 for MTM and 05/53973-0), CNPq (BPP 300351/2008-9 to MAMS) FTV was a Master’s student supported by CAPES.+ Corresponding author: [email protected] 23 April 2010Accepted 4 November 2010

    Intragenomic variation in the second internal transcribed spacer of the ribosomal DNA of species of the genera

    Culex and Lutzia (Diptera: Culicidae)

    Fabiana Tavares Vesgueiro1, Bruna Demari-Silva1, Rosely dos Santos Malafronte2, Maria Anice Mureb Sallum1, Mauro Toledo Marrelli1/+

    1Departamento de Epidemiologia, Faculdade de Saúde Pública 2Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, São Paulo, SP, Brasil

    Culex is the largest genus of Culicini and includes vectors of several arboviruses and filarial worms. Many species of Culex are morphologically similar, which makes their identification difficult, particularly when using female speci-mens. To aid evolutionary studies and species distinction, molecular techniques are often used. Sequences of the sec-ond internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) from 16 species of the genus Culex and one of Lutzia were used to assess their genomic variability and to verify their applicability in the phylogenetic analysis of the group. The distance matrix (uncorrected p-distance) that was obtained revealed intragenomic and intraspecific variation. Because of the intragenomic variability, we selected ITS2 copies for use in distance analyses based on their second-ary structures. Neighbour-joining topology was obtained with an uncorrected p-distance. Despite the heterogeneity observed, individuals of the same species were grouped together and correlated with the current, morphology-based classification, thereby showing that ITS2 is an appropriate marker to be used in the taxonomy of Culex.

    Key words: Diptera - Culex - ITS2 - rDNA - phylogeny - concerted evolution

  • ITS2 variation in Culex and Lut. bigoti • Fabiana Tavares Vesgueiro et al.2

    to the concerted evolution mechanism, multiple cop-ies of rDNA tend to homogenize, maintaining a low level of intraspecific and intragenomic variation. Thus, rDNA transcribed spaces (ITS) sequences prove use-ful for solving evolutionary relationships at different taxonomic levels, including recently diverged taxa. It is also helpful for solving problems associated with the identification of morphologically similar species by polymerase chain reaction (PCR) (Collins & Paskewitz 1996, Djadid et al. 2007). The first molecular approach for investigating the phylogenetic relationships between 14 species of four subgenera of the Culex genus was carried out by Miller et al. (1996), using both ITS1 and ITS2 rDNA. The authors found sequence heterogeneity for both ITSs between species and, in some cases, be-tween individual mosquitoes. Importantly, among the species included in Miller et al. (1996), two were ana-lyzed in our study. Furthermore, a phylogenetic analy-sis of the Vomerifer and Pedroi groups of the Spissipes Section of Culex (Melanoconion) also employed ITS2 sequences (Navarro & Weaver 2004).

    To gain a better understanding of group relation-ships, we investigated the intragenomic, intraspecific and interspecific variation among 16 mosquito species of the Culex genus, including those belonging to the subgenera, Culex, Microculex Theobald, Melanocon-ion, Phenacomyia Harbach and Peyton and one taxon of the genus Lutzia. The main objective of this study was to establish the taxonomic relationships within the Culex genus in the neotropics, in order to test the phy-logenetic placement of the subgenera Phenacomyia and Lutzia relative to Culex.

    MATeRIAlS AND MeThoDS

    Mosquitoes - The species sampled for this study, specimen abbreviations, details about field collections and GenBank/EMBL clone accessions are shown in Supplementary data. The mosquitoes were collected from 2006-2008 in 14 localities in the four following Brazilian states: São Paulo (SP), Espírito Santo (ES), Rio Grande do Sul and Paraná. Most species were col-lected in their immature larval or pupal stage, though some adults were captured in the field. The immature specimens were reared in the laboratory to the adult stage. Male genitalia, larval and/or pupal exuviae, or all three, were mounted on Canada balsam on microscope slides and deposited for record purposes in the Entomo-logical Collection of the School of Public Health of the University of São Paulo, Brazil. ITS2 sequences were characterized from adult males.

    DNA extraction and amplification - Genomic DNA was extracted from specimens that were initially pre-served in 100% ethanol at room temperature in the field and subsequently frozen at -80ºC. DNA was extracted from the specimens following the tissue DNA extraction protocol provided with the DNeasy Blood & Tissue Kit (QIAGEN, Crawley, UK). The DNA extracted from all samples was preserved at -80ºC for future use. The PCR amplification of the ITS2 region was carried out using the primers 5.8SF (5’-ATCACTCGGCTCGTGGATCG-3’)

    and 28SR (5’-ATGCTTAAATTTAGGGGGTAGTC-3’) (Djadid et al. 2007). PCR products were amplified in a 25 µL reaction mix containing 3-10 µL of genomic DNA (1st elution), 2.5 µL of 10X PCR buffer (Fermentas), 1.25 mM MgCl2, 0.12 mM of each dNTP (Fermentas), 10 pi-comols of each primer, 8% dimethyl sulfoxide, 1.25 U of Taq DNA polymerase (Fermentas) and [DNase/RNase-free distilled water (Invitrogen)]. PCRs were performed at 94ºC for 2 min, 34 cycles at 94ºC, 55ºC and 72ºC for 30 s, followed by a final extension step at 72ºC for 20 min. PCR products were visualized by ethidium bromide staining after electrophoresis in a 1.5% agarose gel. The products were purified using isopropanol and electrophoresed again to quantify DNA.

    DNA cloning and sequencing - Amplicons contain-ing the ITS2 region and some of the flanking 5.8S and 28S genes were cloned using the InsTAcloneTM PCR Cloning Kit (Fermentas) and Escherichia coli strain DH5α (Invitrogen) for transformation. The cloned se-quences were extracted by PCR colony using the same protocol as ITS2 PCR, but using vector plasmid prim-ers M13/pUC F (5’-GTAAAACGACGGCCAGT-3’) and M13/pUC R (5’-CAGGAAACAGCTATGAC-3’). The clones were sequenced in both directions, using the same vector plasmid primers with a Big Dye® Ter-minator version 3.1 Cycle Sequencing Kit (PE Applied Biosystems, Warrington, England) in an ABI PRISM® 3100 automated sequencer (Genetic Analyzer/HITACH, Applied Biosystems, Foster City, CA, USA).

    Sequence analysis - All ITS2 sequences obtained, including parts of the 5.8S and 28S genes, were ana-lyzed and edited using Chromas Lite version 2.01 soft-ware (Technelysium Pty Ltd 2007). The ITS2 sequenc-es obtained were compared with sequences available in the GenBank/EMBL database, using the Basic Lo-cal Alignment Search Tool (Zhang et al. 2000). The ITS2 sequences were annotated using the ITS2 anno-tation tool (Keller et al. 2009). Nucleotide sequences were aligned using the multiple alignment program CLUSTALX version 2.0 (Larkin et al. 2007) with a gap opening penalty of 10 and a gap extension penalty of 5. ITS2 sequences of three species available in Gen-Bank were used as an out-group, including Psoropho-ra (Janthinosoma) ferox (Von Humboldt) (M95129), Aedes (Aedimorphus) vexans Meigen (EF539857) and Aedes (Stegomyia) aegypti Linnaeus (AY512670). Be-cause of the large interspecific variability, we edited the alignment using the program MacClade version 4.3 (Maddison & Maddison 2000). We used the proposed ITS secondary structure to minimize ambiguities in the sequence alignments.

    Selection of clones by secondary structure - At least two sequences of each individual were used to obtain the secondary structures with the Model tool available in the ITS2 database (Schultz et al. 2006, Selig et al. 2008). Given the interspecific variability in the spatial pattern from structures, especially between different subgenera, we clustered one sequence of each species with known structures with three sequences of each

  • 3Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(1), February 2011

    species with unknown structures. We then checked each cluster for the presence of sequences with assigned structures. Thus, the assigned structure that resulted in the best alignment with the template sequences was selected as a model for transferring the structure to the homologous sequences within all 144 clones. To ensure high prediction performance, the structure was trans-ferred only when more than 75% of all base pairs (bp) of each helix could be used. Only clones with a high-quality model (> 75%) were selected.

    Sequence alignment and distance analysis - Se-lected sequences were aligned in CLUSTALX version 2.0 (Larkin et al. 2007). The alignment of the ITS2 sequences, excluding 5.8S and 28S, was edited manu-ally in MacClade version 4.0 (Maddison & Maddison 2000). The divergences between the ITS2 sequences were accessed by distance matrices, produced in Mo-lecular Evolutionary Genetics Analysis version 4.0 (Ta-mura et al. 2007). A neighbour-joining (NJ) (Saitou & Nei 1987) topology was generated with an uncorrected p-distance in Phylogenetic Analysis Using Parsimony* (PAUP*) version 4.0 (Swofford 2003). Statistical sup-port for the branches was estimated by bootstrap analy-sis with 1,000 replicates.

    ReSulTS

    We sequenced ITS2 PCR products from 31 individu-als belonging to the subgenera Microculex, Melanoco-nion, Culex and Phenocomyia and one of the genus Lutzia (Supplementary data). The number of clones sequenced per individual ranged from 3-7, with 144 sequences obtained in total. An alignment of 93 dif-ferent sequences is shown in Supplementary data. The length of ITS2 fragments, including 41 bp of the final portion of the ribosomal 5.8S gene and 41 bp of the initial portion of the 28S of rDNA, ranged from 281-421 bp in Culex (Microculex) imitator Theobald (imit 1) and Cux. (Cux.) quinquefasciatus (quin 1), respec-tively. The GenBank/EMBL accessions are shown in Supplementary data.

    Intragenomic variation - Of the 31 specimens ana-lyzed only nine (moll 2, bidens, nigri 2, decl 1, dyius, ybarmis, caud 1, bigoti 1 and bigoti 2) showed no varia-tion in the length of cloned fragments within each in-dividual. However, of these, only clones derived from Culex (Cux.) mollis Dyar and Knab (moll 2) showed no intragenomic variation. Differences in sequence length between clones were mainly due to insertions or de-letions (indels) in ITS2 microsatellite repeat regions. There were no indels in either 5.8S or 28S. However, in the ITS2, clones from Culex (Cux.) dolosus Lynch Arribalzaga (dolo 2) showed differences of up to 16 bp. Details regarding the number of clones sequenced, the variants from each individual selected using the sec-ondary structure, the length of ITS2 only (excluding the 5.8S and 28S flanking regions) and the mean uncorrect-ed p-distance between clones are shown in Table I.

    Intraspecific variation - Details of intraspecific variation, the number of specimens per species, the length of the ITS2 region (excluding the flanking re-gions 5.8S and 28S), the number of clones selected using the secondary structure, the mean uncorrected p-distance and the number of variants of each species are shown in Table II. Lutzia (Lut.) bigoti had the low-est average divergence of all selected sequences (0.3% for all clones, 0.4% for variants) and Cux. (Mcx.) imita-tor had the highest average divergence (3.8% for all of the selected clones, 5% for variants). In the distance matrix, with uncorrected p-distance made by select-ing and grouping only variations per individual, there was greater divergence (7.1%) between the clones of specimens of Cux. (Mcx.) imitator from SP (imit 2) and ES (imit 1). Culex (Mel.) caudelli (caud 1 and caud 2) showed a distance of 1.4% and the other distances be-tween individuals of the same species were below 0.7% (distance matrix not shown).

    Interspecific variation - Considering only the ITS2 region, the species of subgenus Microculex showed the shortest length sequences (199-213 bp), while species of subgenus Culex presented the largest fragments (277-339 bp). The length ranged from 205-213 bp in Melanoconion, from 268-276 bp in Phenacomyia and from 276-278 bp in Lut. bigoti. The range of guanine-cytosine contents of ITS2 variants selected by secondary structure was as fol-lows: subgenus Microculex (50.2-50.3%), Melanoconion (45.4-50.9%), Culex (52.8-57.5%), Phenacomyia (50.6-53%) and Lutzia (55.4-55.8%). According to the data from the distance matrix with uncorrected p-distance between variants of each species (matrix not shown), the shortest distance (0.5%) in different species was ob-served between Cux. (Cux.) declarator Dyar and Knab and Culex (Cux.) bidens Dyar and between Cux. (Cux.) coronator and Culex (Cux.) usquatus. In the subgenus Melanoconion, the shortest distance was between Culex (Mel.) ybarmis and Culex (Mel.) aliciae (0.9%).

    NJ topology - The identification of homologous sites and manual editing of the sequence alignment was diffi-cult because there were many indels and nucleotide sub-stitutions in the sequences. It was therefore not possible to produce a phylogenetic tree by the Maximum Parsi-mony method, using PAUP* version 4.0. Consequently, only the NJ topology was generated to examine the vari-ability between cloned sequences.

    In NJ topology, within the subgenus Culex, Culex nigripalpus + Culex chidesteri Dyar, Cux. declarator + Cux. bidens and Cux. mollis + Cux. dolosus formed well-supported groups by bootstrap values, which were 100% for the first two groups and 97% for the latter. The cluster of complex coronator species (Cux. usquatus and Cux. coronator) was also supported by a high bootstrap value (100%). Only Cux. quinquefasciatus was not grouped with all other taxa of the subgenus, occupying a position as a neighbouring group to the others. Lutzia and the sub-genus, Phenacomyia, were placed within the Culex sub-genus. Subgenera Melanoconion and Microculex formed two distinct but closely related groups.

  • ITS2 variation in Culex and Lut. bigoti • Fabiana Tavares Vesgueiro et al.4

    DISCuSSIoN

    According to the current classification of the Culi-cine, this tribe includes four genera, Culex, Deinocerites, Galindomyia and Lutzia. The latter was previously con-sidered a subgenus of the genus Culex (Bram 1967) and was elevated to genus by Tanaka (2003). It is interesting

    to note that in the NJ topology, Lut. bigoti was recovered within the genus Culex (Figure), which thus conflicts with Tanaka’s (2003) classification proposal. Navarro and Liria (2000) inferred the phylogenetic relationships between seven subgenera of Culex and Deinocerites by parsimony analysis of the characteristics of the fourth instar larval mouthparts. Parsimony topologies showed

    TABLE IIntragenomic variability between the internal transcribed sequences (ITS2) sequences of all clones and variants,

    both selected by the secondary structure in the same individual

    SpecimensITS2 length

    (bp)

    All clones selected Variants selected

    Clones(n)

    Mean uncorrectedp-distance

    Variants(n)

    Mean uncorrectedp-distance

    coro 1coro 2coro 3usqua 1usqua 2moll 1moll 2bidens 1chid 1dolo 1dolo 2nigri 1nigr 2quin 1quin 2quin 3quin 4decl 1imit 1imit 2zete 1aliciae 1dyius 1 ybarmis 1caud 1caud 2corni 1corni 2bigoti 1bigot 2

    304-302298-304300-306294-297294-298281-285

    292277

    286-288283-285268-284277-280

    280336-339324-335332-338333-336283-287

    199211-213206-207208-212

    205218205205279

    268-276276278

    635555545565554653255353544343

    0.0190.0020.0220.0070.0070.009

    00.0040.0090.0110.0110.0140.0020.0070.0110.0090.0090.014

    00

    0.0020.0160.0060.0090.0290.0200.0020.0070.0020.002

    525524134225243422123322422222

    0.0230.0030.0240.0070.0090.007

    00.0050.0090.0180.0190.0140.0040.0090.0150.0130.0150.021

    00

    0.0030.0160.0100.0140.0320.0400.0040.0110.0040.004

    bp: base pairs.

  • 5Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(1), February 2011

    Lutzia in a basal position sharing a sister-group rela-tionship with the clade that includes species of Culex. This result confirmed the classification of Belkin (1962), which treated Lutzia as a specialized lineage that shares a similarity and therefore ancestry with members of the subgenus Culex. Belkin also corroborated the topology obtained by Miller et al. (1996) from their analysis of rDNA sequences. Nevertheless, the conflicting posi-tion of Lutzia presented in our study was supported by topologies generated using sequences of the mitochon-drial cytochrome oxidase (COI) gene (Demari-Silva et al. 2011). The results of our molecular analysis employ-ing the ITS2 and the COI sequence data do not support a genus position for Lutzia. The NJ topology (Figure) recovered the subgenus Phenacomyia embedded within a group that includes members of the subgenus Culex (90% bootstrap support).

    As with Lutzia, our results do not support the cur-rent status of Phenacomyia as a subgenus of Culex, as was proposed by Harbach and Peyton (1992). The placement of Phenacomyia within the subgenus Culex also disagrees with the topology of Navarro and Liria (2000), in which Phenacomyia is placed in a basal po-sition relative to the clade that includes species of the

    subgenus Culex. Again, analyses of the ITS2 sequence data were corroborated by analysis of the COI mtDNA (Demari-Silva et al. 2011). Furthermore, the results of the ITS2 sequence data showed that in the current clas-sification, the subgenus Culex is paraphyletic because it excludes Phenacomyia.

    Species of the subgenera Microculex and Melanoco-nion formed a monophyletic clade (96% bootstrap sup-port) and these subgenera are clustered in two distinct groups, each supported by a 100% bootstrap analysis value (Figure). The results of the NJ analysis are in agreement with the current classification of Culex, with individuals of the same species and species complex grouped together. Considering the interspecific varia-tion in ITS2 sequences, species belonging to the Cux. coronator (Cux. usquatus and Cux. coronator) complex formed a clade (Figure) and species with similar mor-phology (Cux. bidens + Cux. declarator and Cux. nigri-palpus + Cux. chidesteri) also clustered. The positioning of Cux. (Cux.) quinquefasciatus as a sister group of the clade, which includes the others species of Culex, is sim-ilar to the topology obtained by Navarro and Liria (2000) based on the morphological characteristics of the larval maxilla and mandibula. The authors analyzed the phy-

    TABLE II

    Intragenomic variability between the internal transcribed sequences (ITS2) sequences of all clones and variants, both selected by the secondary structure in the same species

    Species(number of specimens)

    ITS2 length(bp)

    All clones selected Variants selected

    Clones(n)

    Mean uncorrectedp-distance

    Variants(n)

    Mean uncorrectedp-distance

    Culex (Cux.) coronator (3)Culex (Cux.) usquatus (2)Culex (Cux.) mollis (2)Culex (Cux.) bidens (1)Culex (Cux.) chidesteri (1)Culex (Cux.) dolosus (2)Culex (Cux.) nigripalpus (2)Culex (Cux.) quinquefasciatus (4)Culex (Cux.) declarator (1)Culex (Mcx.) imitator (2)Culex (Mel.) zeteki (1)Culex (Mel.) aliciae (1)Culex (Mel.) dyius (1)Culex (Mel.) ybarmis (1)Culex (Mel.) caudelli (2)Culex (Phc.) corniger (2)Lutzia (Lut.) bigoti (2)

    298-306294-298281-285

    277286-288268-285277-280324-339283-287199-213206-207208-212

    205218205

    268-276276-278

    14101045111020375353977

    0.0190.0070.0120.0040.0090.0170.0090.0090.0140.0380.0020.0160.0060.0090.0270.0060.003

    1275344713233322644

    0.0190.0100.0110.0050.0090.0200.0120.0100.0210.0500.0030.0160.0100.0140.0300.0080.00

    bp: base pairs.

  • ITS2 variation in Culex and Lut. bigoti • Fabiana Tavares Vesgueiro et al.6

    logenetic relationships of 18 species from eight genera of the tribe Culicine, including Cux. quinquefasciatus, Cux. dolosus, Cux. nigripalpus and Cux. coronator for subgenus Culex. In the clade formed by species of these subgenera, Cux. quinquefasciatus is a sister of a clade that includes Cux. dolosus, Cux. nigripalpus and Cux. coronator, which is consistent with the ITS2 NJ topol-ogy analysis. Cux. ybarmis and Cux. aliciae of the sub-

    genus Melanoconion showed the lowest mean p-distance between their clones (0.9%). Due to the great morpho-logical similarity and consequent difficulty in differen-tiating Cux. aliciae from Culex dyius, we expected to find that these two species were related at the molecular level. In this subgenus, dissection and examination of the lateral plate of the aedeagus from a lateral perspec-tive is essential for species identification (Rozeboom

    Distance tree generated with the variants selected by internal transcribed sequences secondary structure (specimen codes-clone numbers). Only the model structures with high-quality (> 75%) in the transfer helices, using “Homology Modeling” tool were selected. The topology was constructed by neighbor-joining method, uncorrected p-distance, bootstrap confidence levels (%) from 1,000 replications and rooted with Aedes vexans (EF539857), Psorophora ferox (M95129) and Aedes aegypti (AY512670) sequences of GenBank/EMBL.

    Ae. aegypti

    Ae. vexansPs. ferox

    quin 3-2

    quin 3-1

    quin 1-2

    quin 4-2

    quin 2-4

    quin 3-3

    quin 1- 4quin 3-3quin 1-1

    quin 2-3quin 2-1

    quin 4-1quin 1-3

    imit 2-1imit 2-2

    imit 1-1zete 1-1zete 1-2

    zete 1-3

    aliciae 1-2aliciae 1-1

    aliciae 1-3

    ybarmis 1-1

    dyius 1-2dyius 1-1

    ybarmis 1-2

    caud 1-1caud 1-4caud 2-1caud 1-2

    caud 1-3caud 2-2

    moll 1-1moll 1-4moll 1-3

    moll 2-1moll 1-2dolo 1-1

    dolo 1-1dolo 2-4

    dolo 2-1bidens 1-1

    bidens 1-2bidens 1-3

    decl 1-1decl 1-2

    nigr 1-2nigr 1-1

    nigr 2-1nigr 1-3

    nigr 1-4nigr 1-5

    chid 1-2chid 1-4

    chid 1-1chid 1-3

    bigoti 2-1bigoti 1-2bigoti 2-2

    bigoti 1-1coro 1-3coro 3-2coro 1-4

    coro 3-1coro 3-5coro 2-2

    coro 2-1usqua 1-2

    usqua 1-3usqua 1-4

    usqua 1-5usqua 1-1

    usqua 2-1

    usqua 2-1

    coro 1-1coro 1-5

    coro 1-6coro 3-3coro 3-4

    corni 1-2corni 1-1

    corni 1-5corni 2-2

    corni 2-1

    100

    100

    100

    100

    100

    96

    92

    100

    90

    100

    100

    100

    100

    100

    100

    99

    100

    97

    100

    83

    61

    56

    63

    Microculex

    Melanoconion

    Culex

    Culex

    Lutzia

    Phenacomyia

    Culex

    Ae. aegypti

    Ae. vexansPs. ferox

    quin 3-2

    quin 3-1

    quin 1-2

    quin 4-2

    quin 2-4

    quin 3-3

    quin 1- 4quin 3-3quin 1-1

    quin 2-3quin 2-1

    quin 4-1quin 1-3

    imit 2-1imit 2-2

    imit 1-1zete 1-1zete 1-2

    zete 1-3

    aliciae 1-2aliciae 1-1

    aliciae 1-3

    ybarmis 1-1

    dyius 1-2dyius 1-1

    ybarmis 1-2

    caud 1-1caud 1-4caud 2-1caud 1-2

    caud 1-3caud 2-2

    moll 1-1moll 1-4moll 1-3

    moll 2-1moll 1-2dolo 1-1

    dolo 1-1dolo 2-4

    dolo 2-1bidens 1-1

    bidens 1-2bidens 1-3

    decl 1-1decl 1-2

    nigr 1-2nigr 1-1

    nigr 2-1nigr 1-3

    nigr 1-4nigr 1-5

    chid 1-2chid 1-4

    chid 1-1chid 1-3

    bigoti 2-1bigoti 1-2bigoti 2-2

    bigoti 1-1coro 1-3coro 3-2coro 1-4

    coro 3-1coro 3-5coro 2-2

    coro 2-1usqua 1-2

    usqua 1-3usqua 1-4

    usqua 1-5usqua 1-1

    usqua 2-1

    usqua 2-1

    coro 1-1coro 1-5

    coro 1-6coro 3-3coro 3-4

    corni 1-2corni 1-1

    corni 1-5corni 2-2

    corni 2-1

    100

    100

    100

    100

    100

    96

    92

    100

    90

    100

    100

    100

    100

    100

    100

    99

    100

    97

    100

    83

    61

    56

    63

    Microculex

    Melanoconion

    Culex

    Culex

    Lutzia

    Phenacomyia

    Culex

  • 7Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 106(1), February 2011

    1950). In Cux. dyius, the lateral plate of the aedeagus is slender with a long, broad, blunt point at an angle in the middle, with a few small teeth at its apex (Lane 1953). In Cux. aliciae, this structure is very similar (Duret 1953). In Cux. ybarmis this structure is different, presenting the apical, dorsal and sterna processes, with its dorsal process curved (Lane 1953).

    Cux. imitator, the only species of Microculex ana-lyzed in this study, proved to have the greatest intraspe-cific divergence (7.1%) among specimens from SP (imit 2) and ES (imit 1) (data not shown). In addition, mor-phological variations were observed in the pupal exuviae of specimens from SP and ES, suggesting that the two individuals may belong to different species. The pupa of an individual of Cux. imitator, from the same location as the specimen from ES that was analyzed molecularly, showed typical characteristics of Cux. imitator, includ-ing a dark spot on segments II, III and IV and the ar-rangement pattern of setae 2, 3, 4 and 5 of segment II. The specimen from SP did not have the spot and had another arrangement pattern of its setae (morphological data not shown). Analysis of COI sequences also indi-cated high intraspecific variation for these specimens (Demari-Silva et al. 2011), thus corroborating the mor-phological hypothesis that these individuals may belong to different taxa of a species complex. In a phylogenetic analysis of species of the Pedroi and Vomerifer groups of Melanoconion, Navarro and Weaver (2004) used ITS se-quence analysis and observed an intraspecific variation of 0-3.3% between individuals from the same location (Culex pedroi in Venezuela or Cux. pedroi-Peru form in Peru) and 0-4.5% between those from different locations (Culex vomerifer: Colombia vs. Peru).

    The intragenomic and intraspecific variability of clones contradicted the theory of concerted evolution and prevented phylogenetic analysis because the alignment that is a fundamental step for reliability of any evolution-ary analysis was compromised. There is a possibility of PCR-induced mutations, which might interfere in the as-sessment of intragenomic and intraspecific variability because the lack of 3’-5’ proofreading ability of the Taq DNA polymerase used in our experiments results in an error rate (mutations per nucleotide per cycle) of approxi-mately 1 in 10,000 bases (Eckert & Kunkel 1991, Barnes 1994). However, considering that PCR errors are also proportionally related to the length of the PCR products and that approximately 50,000 bases were sequenced in the present study, the interference of PCR-induced muta-tions in our final conclusions was not significant.

    Some aspects of the phenomenon of intragenomic variability remains unclear, but the high intragenomic heterogeneity observed seems to be the result of a com-parison between sequences of different clusters undergo-ing different levels of evolution. Similar results were ob-served by Miller et al. (1996), who showed heterogeneity between sequences of the same individual in different populations of the Culex pipiens complex, thus invali-dating the use of ITS markers for phylogenetic analysis of taxa that exhibit intragenomic variation. However, there was general agreement between the groupings ob-

    tained in the topologies for species of the genus Culex and the recognition of taxa by morphology. Therefore, in both this study and the previously mentioned work, the trees obtained generally corroborated the traditional taxonomy of taxa, indicating that despite the presence of intragenomic variation, the ITS2 marker seems to be ap-propriate for solving taxonomic doubts in the group.

    ReFeReNCeS

    Barnes WM 1994. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci USA 91: 2216-2220.

    Belkin JN 1962. The mosquitoes of the South Pacific (Diptera, Cu-licidae), vol. I, University of California Press, Berkeley and Los Angeles, 545 pp.

    Bram RA 1967. Classification of Culex subgenus Culex in the New World (Diptera, Culicidae). Proc US Nat Hist Mus 120: 1-122.

    Collins FH, Paskewitz SM 1996. A review of the use of ribosomal DNA (rDNA) to differentiate among cryptic Anopheles species. Insect Mol Biol 5: 1-9.

    Demari-Silva B, Vesgueiro FT, Sallum MA, Marrelli MT 2011. Taxo-nomic and phylogenetic relationships between species of the genus Culex (Diptera: Culicidae) from Brazil inferred from the Cyto-chrome C Oxidase I mitochondrial gene. J Med Entomol, in press.

    Djadid ND, Gholizadeh S, Tafsiri E, Romi R, Gordeev M, Zakeri S 2007. Molecular identification of Paleartic members of Anopheles maculipennis in northern Iran. Malar J 6: 6.

    Duret JP 1953. Las especies argentinas de Culex (Melanoconion) (Diptera: Culicidae). Rev Soc Entomol Argent 16: 67-76.

    Eckert KA, Kunkel TA 1991. DNA polymerase fidelity and the polymerase chain reaction. PCR Methods Appl 1: 17-24.

    Fontes G, Braun RF, Fraiha Neto H, Vieira JB, Padilha SS, Rocha RC, da Rocha EM 2005. Filariose linfática em Belém, estado do Pará, Norte do Brasil e a perspectiva de eliminação. Rev Soc Bras Med Trop 38: 131-136.

    Fontes G, Rocha EM, Brito AC, Antunes CM 1998. Lymphatic filariasis in Brazilian urban area (Maceió, Alagoas). Mem Inst Oswaldo Cruz 93: 705-710.

    Forattini OP 2002. Culicidologia médica: identificação, biologia e epidemiologia, vol. II, EDUSP, São Paulo, 864 pp.

    Harbach RE 2011. Mosquito taxonomic inventory. Culicidae clas-sification. Natural History Museum, London, UK [cited 2009 Nov 11]. Available from: http://mosquito-taxonomic-inventory.info/em/taxonomy/term/6165.

    Harbach RE, Peyton EL 1992. A new subgenus of Culex in the Neo-tropical region (Diptera: Culicidae). Mosq Syst 24: 242-252.

    Humeres SG, Almirón WR, Sabattini MS, Gardenal CN 1998. Es-timation of genetic divergence and gene flow between Culex pipiens and Culex quinquefasciatus (Diptera: Culicidae) in Ar-gentina. Mem Inst Oswaldo Cruz 93: 57-62.

    Keller A, Schleicher T, Schultz J, Müller T, Dandekar T, Wolf M 2009. 5.8S-28S rRNA interaction and HMM-based ITS2 anno-tation. Gene 430: 50-57.

    Knight JW, Nayar JK 2004. Identification of four common Culex (Culex) (Diptera: Culicidae) species from Florida with isoen-zyme analysis. Fla Entomol 87: 1-5.

    Lane J 1953. Neotropical Culicidae, vol. I, EDUSP, São Paulo, 558 pp.

    Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan

  • ITS2 variation in Culex and Lut. bigoti • Fabiana Tavares Vesgueiro et al.8

    PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG 2007. Clustal W and CLUSTALX version 2.0. Bioinformatics 23: 2947-2948.

    Maddison DR, Maddison WP 2000. MacClade 4: analysis of phy-logeny and character evolution, Sinauer, Sunderland, 398 pp.

    Marrelli MT, Floeter-Winter LM, Malafronte RS, Tadei WP, Louren-ço-de-Oliveira R, Flores-Mendoza C, Marinotti O 2005. Ama-zonian malaria vector anopheline relationships interpreted from ITS2 rDNA sequences. Med Vet Entomol 19: 208-218.

    Melo AL, Soccol CR, Thomaz-Soccol V, Nogueira M Jr 2009. Evaluation of Bacillus sphaericus bioinsecticide produced with white soybean meal as culture medium for the control of Culex (Culex) quinquefasciatus. Cad Saude Publica 25: 563-569.

    Miller BR, Crabtree MB, Savage HM 1996. Phylogeny of fourteen Culex mosquito species, including the Culex pipiens complex, inferred from the internal transcribed spacers of ribosomal DNA. Insect Mol Biol 5: 93-107.

    Navarro JC, Liria J 2000. Phylogenetic relationships among eigh-teen neotropical Culicini species. J Am Mosq Control Assoc 16: 75-85.

    Navarro JC, Weaver SC 2004. Molecular phylogeny of the Vomeri-fer and Pedroi groups in the Spissipes section of the subgenus Culex (Melanoconion). J Med Entomol 41: 575-581.

    Regis L, Silva-Filha MHNL, de Oliveira CMF, Rios EM, da Silva SB, Furtado AF 1995. Integrated control measures against Culex quinquefasciatus, the vector of filariasis in Recife. Mem Inst Oswaldo Cruz 90: 115-119.

    Rey JR, O’Meara GF, O’Connell SM, Darsie RF 2006. Variation in the number and position of siphonal setae in Culex quinque-fasciatus from Key West and Vero Beach, Florida, USA. J Am Mosq Control Assoc 22: 355-357.

    Rozeboom LE 1950. A review of the species of Culex of the subge-nus Melanoconion (Diptera: Culicidae). Ann Entomol Soc Am 43: 75-114.

    Saitou N, Nei M 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406-425.

    Sallum MAM, Schultz TR, Foster PG, Aronstein K, Wirtz RA, Wilkerson RC 2002. Phylogeny of Anophelinae (Diptera: Cu-licidae) based on nuclear ribosomal and mitochondrial DNA sequences. Syst Entomol 27: 361-382.

    Schultz J, Müller T, Achtziger M, Seibel PN, Dandekar T, Wolf M 2006. The internal transcribed spacer 2 database: a web server for (not only) low level phylogenetic analyses. Nucleic Acids Res 34: 704-707.

    Selig C, Wolf M, Müller T, Dandekar T, Schultz J 2008. The ITS2 Database II: homology modelling RNA structure for molecular systematics. Nucleic Acids Res 36: 377-380.

    Smith JL, Fonseca DM 2004. Rapid assays for identification of members of the Culex (Culex) pipiens complex, their hybrids, and other sibling species (Diptera: Culicidae). Am J Trop Med Hyg 70: 339-345.

    Swofford DL 2003. PAUP*. Phylogenetic analysis using parsimony (*and other methods), version 4.01b, Sinauer Associates, Sun-derland, Massachusets.

    Tamura K, Dudley J, Nei M, Kumar S 2007. MEGA 4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596-1599.

    Tanaka K 2003. Studies on the pupal mosquitoes of Japan. Genus Lutzia, with establishment of two new subgenera, Metalutzia and Insulalutzia (Diptera, Culicidae). Syst Entomol 9: 159-169.

    Technelysium Pty Ltd 2007. [cited 2009 Oct 10]. Chromas lite ver-sion 2.01. Available from: http://www.technelysium.com.au/chromas_lite.html.

    Turell MJ, O’Guinn ML, Dohm DJ, Jones JW 2001. Vector compe-tence of North American mosquitoes (Diptera: Culicidae) for West Nile Virus. J Med Entomol 38: 130-134.

    Vasconcelos PFC, Travassos-da-Rosa JFS, Travassos-da-Rosa APA, Dégallier N, Pinheiro FP, Sá-Filho GC 1991. Epidemiologia das encefalites por arbovírus na Amazônia brasileira. Rev Inst Med Trop Sao Paulo 33: 465-476.

    Walton C, Sharpe RG, Pritchard SJ, Thelwell NJ, Butlin RK 1999. Molec-ular identification of mosquito species. Biol J Linn Soc 68: 241-256.

    Weaver SC, Ferro C, Barrera R, Boshell J, Navarro JC 2004. Venezu-elan equine encephalitis. Annu Rev Entomol 49: 141-174.

    Williams MR, Savage HM 2009. Identification of Culex (Melanoco-nion) species of the United States using female cibarial armature (Diptera: Culicidae). J Med Entomol 46: 745-752.

    Zhang Z, Schwartz S, Wagner L, Miller W 2000. A greedy algorithm for aligning DNA sequences. J Comput Biol 7: 203-214.

  • 1Supplementary data ITS2 variation in Culex and Lu. bigoti • Fabiana Tavares Vesgueiro et al.

    TABLESpecies studied, specimen codes, localities, geographical coordinates and GenBank/EMBL accessions of clones

    Species Specimens Sex Localities Coordinates GenBank accessions

    Culex (Cux.) coronator Dyar and Knab coro 1 ♂ Campos do Jordão (SP) 22º45’50’’S 45o30’87’’W GU299736-GU299740coro 2 ♂ Linhares (ES) 19o20.917’S 40o07.103’W GU299741-GU299742coro 3 ♂ Aparecida do Norte (SP) 22o50’34.4”S 45o14’45”W GU299743-GU299747

    Culex (Cux.) usquatus Dyar usqua 1 ♂ Pariquera-Açu (SP) 24o43.975’S 47o49.502’W GU299731-GU299735usqua 2 ♂ Pariquera-Açu (SP) 24o43.975’S 47o49.502’W GU299729-GU299730

    Culex (Cux.) mollis Dyar and Knab moll 1 ♂ Pariquera-Açu (SP) 24o43.975’S 47o49.502’W GU299706-GU299709moll 2 ♂ Pariquera-Açu (SP) 24o43.975’S 47o49.502’W GU299705

    Culex (Cux.) chidesteri Dyar chid 1 ♂ Pindamonhangaba (SP) 22o57’35.1”S 45o27’08.6”W GU299725-GU299728

    Culex (Cux.) dolosus Lynch Arribalzaga dolo 1 ♂ Pindamonhangaba (SP) 22o57’35.1”S 45o27’08.6”W GU299710-GU299711dolo 2 ♂ Pindamonhangaba (SP) 22o57’35.1”S 45o27’08.6”W GU299712-GU299713

    Culex (Cux.) nigripalpus Theobald nigri 1 ♂ Pariquera-Açu (SP) 24o43.975’S 47o49.502’W GU299719-GU299723nigri 2 ♂ Pariquera-Açu (SP) 24o43.975’S 47o49.502’W GU299724

    Culex (Cux.) quinquefasciatus Say quin 1 ♀ Pariquera-Açu (SP) 24o43.975’S 47o49.502’W GU299748-GU299751quin 2 ♂ Pariquera-Açu (SP) 24o43.975’S 47o49.502’W GU299752-GU299754quin 3 ♂ Bento Gonçalves (RS) 29o09’28.90”S 51o30’43.55”W GU299755-GU299758quin 4 ♂ Bento Gonçalves (RS) 29o09’28.90”S 51o30’43.55”W GU299759-GU299760

    Culex (Cux.) declarator Dyar and Knab decl 1 ♂ Pariquera-Açu (SP) 24o43.975’S 47o49.502’W GU299717-GU299718

    Culex (Cux.) bidens Dyar bidens 1 ♂ Aparecida do Norte (SP) 22o50’34.4”S 45o14’45”W GU299714-GU299716

    Culex (Mcx.) imitator Theobald imit 1 ♂ Santa Teresa (ES) 19o55’54.46”S 40o35’41.61”W GU299677imit 2 ♂ Pindamonhangaba (SP) 22o57’35.1”S 45o27’08.6”W GU299678-GU299679

    Culex (Mel.) zeteki Dyar zetk 1 ♀ Pariquera-Açu (SP) 24o43.975’S 47o49.502’W GU299680-GU299682

    Culex (Mel.) aliciae Duret aliciae 1 ♂ Pariquera-Açu (SP) 24o43.975’S 47o49.502’W GU299683-GU299685

    Culex (Mel.) dyius Root dyius 1 ♂ Linhares (ES) 19o20.917’S 40o07.103’W GU299686-GU299687

    Culex (Mel.) ybarmis Dyar ybarmis 1 ♂ Linhares (ES) 19o20.917’S 40o07.103’W GU299688-GU299689

    Culex (Mel.) caudelli (Dyar & Knab) caud 1 ♂ Pariquera-Açu (SP) 24o43.975’S 47o49.502’W GU299690-GU299693caud 2 ♂ Pariquera-Açu (SP) 24o43.975’S 47o49.502’W GU299694-GU299695

    Culex (Phc.) corniger Theobald corni 1 ♂ Pariquera-Açu (SP) 24o43.975’S 47o49.502’W GU299700-GU299702corni 2 ♂ Pariquera-Açu (SP) 24o43.975’S 47o49.502’W GU299703-GU299704

    Lutzia (Lut.) bigoti (Bellardi) bigoti 1 ♀ São Paulo (SP) 23o26’12.44”S 46o38’05.09”W GU299696-GU299697bigoti 2 ♀ S. José dos Pinhais (PR) 25o33’14.48”S 49o14’08.69”W GU299698-GU299699

    ES: Espírito Santo; PR: Paraná; RS: Rio Grande do Sul; SP: São Paulo.